Prions cause fatal, incurable, infectious neurodegenerative diseases in animals and humans. Transmission occurs by corruption of cellular prion protein (PrPC) by the pathogenic conformation, PrPSc. Prions share with nucleic acid based pathogens, the ability to propagate strain information. Our overarching goal is to understand the biological and physico-chemical basis of prion strain variation, and how strains mutate and acquire fitness in the absence of nucleic acids, phenomena that are linked to their underlying pathogenesis and zoonotic potential. Our focus remains largely on chronic wasting disease (CWD), a burgeoning, and highly contagious prion disorder of captive and wild cervids. The discoveries of distinct CWD strains, and of prions in deer skeletal muscle and elk antler velvet, have unmistakable relevance to human health. The currently indistinguishable PrPSc conformations and unstable transitions of CWD strains are consistent with their separation by relatively low energy barriers. Strain stability also differs between deer and elk. Our overarching hypothesis is that host PrPC primary structure influences the portfolio of thermodynamically preferred PrPSc conformations that are kinetically selected during prion propagation. The following Aims comprise our research design and methods: 1: To produce gene targeted (Gt) mice, in which the mouse Prnp coding sequence is substituted by that of deer or elk. This will enable us to explore the hypothesis that residue 226, the sole difference in deer and elk PrP primary structures, resides within a critical domain influencing PrP conversion and strain stability. 2: Gt mice provide a resource to precisely characterize the properties of CWD strains, and to assess the contributions of PrP primary structure on strain stability. Transgenetic studies of the domain encompassing cervid residue 226 will also be extended in this Aim. 3: We will use Gt mice and existing transgenic models to assess whether CWD strains interfere or synergize during disease, and to explore peripheral pathogenesis of strains, which are likely factors in the unparalleled contagious transmission of CWD. 4: We will develop innovative cell culture resources for quantifying CWD prions and characterizing strain properties. We hypothesize that prion strains are subject to selective pressure, and we will assess the effects of modifiers of prion replication on the formation of strain variants with altered biological and biochemical properties of cervid PrPSc. 5: Finally, we hypothesize that the instabilities of CWD strains, and their currently indiscernible biochemical properties, reflect thermodynamic similarities between PrPSc constituting these strains. We will examine whether purified strains differ with respect to protease-sensitive PrPSc, and assess the sensitivity of Conformational Dependent Immunoassay, Fourier-transform infrared spectroscopy, luminescent conjugated polymers, and Protein Misfolding Cyclic Amplification approaches for strain discrimination. We anticipate that our work will continue to have a sustained, powerful impact on this field, and likely related diseases involving protein misfolding, as well as clarifying many remaining enigmatic aspects of CWD biology.